Animal models are critical for the preclinical evaluation of gene therapeutic strategies. The SCID-hu mouse, harboring a normally functioning human thymus, sustains thymopoiesis for as long as one year and is susceptible to HIV infection. We and others have developed this system further for exogenous stem cell transfer. Purified CD34+ hematopoletic progenitor cells when gene transduced with retroviral vectors and introduced into thymic grafts, develop into normal human T lymphocytes. We have also recently demonstrated that vector delivered anti-HIV gene therapeutic constructs such as ribozymes were retained and expressed in these cells as they mature. The combination of virus-induced cell depletion and gene transduced CD34+ cell reconstitution taking place in a relatively short time, provides us with a unique experimental system in which to address many critical issues relevant for the success of gene therapy approaches. Furthermore, at present, the SCID-hu system is the only in vivo system to accurately evaluate the thymopoietic potential and HIV resistance of vector transduced hematopoietic progenitor cells. Several new exciting developments occurred recently in the areas of stem cell biology, lentiviral gene transfer vectors, ribozyme targeting, and RNA-based therapeutics and, therefore, the stage is currently set to achieve success. In the present proposal, we would like to exploit these new technologies and build upon our recent progress. Experiments outlined here (Project 2) are interactive and complimentary to the objectives of accompanying interactive R01 proposal (Project 1) by J. Rossi entitled "Combinatorial use of anti-HIV RNA-based therapeutics." The specific objectives of our proposal are: l) Determine the effect of retrovirally transduced pol III promoter driven nucleolar, nuclear and cytoplasmtargeted anti-HIV nbozymes TAR and RBE decoys, either individually or in combination, on the lineage specific differentiation of CD34+ cells into macrophages in vitro and into thymocytes in vivo in the SCID-hu thy/liv grafts and investigate the mechanism of action of RNA-based therapeutics in differentiated cells. 2) Determine the in vivo protective effects of different anti-HIV-1 RNAs, individually and in combination, in SCID-hu mice thy/liv grafts after HIV-1 challenge. 3) Determine the ability of new generation SIV-based lentiviral vectors to transduce various hematopoietic precursor cells that include CD34+ cells and the newly described primitive hematopoietic cells, namely CD34+ and KDR+ precursor cells, as well as side population (SP) cells. 4) Determine the engraftment and thymopoietic potential of lentivirally transduced primitive hematopoietic precursor cells i.e., a) CD34+ and KDR+ cells, b) SP cells in the SCID-hu mouse thymic microenvironment.